Download Spectrophotometer

Spectrophotometer, Installation
Maintenance and Repair
Prof. Moustafa Moustafa Mohamed
Spectrophotometer
• The word spectrophotometer is derived from the Latin
word spectrum, which means image, and the Greek
word phos or photos, which means light.
• Generally, light from a lamp with special characteristics
is guided through a device, which selects and separates
a determined wave length and makes it pass through a
sample.
• The light intensity leaving the sample is captured and
compared with that which passed through the sample.
Transmittance, which depends on factors such as the
substance concentration is calculated from this
intensity ratio.
PURPOSE OF THE EQUIPMENT
• The spectrophotometer is used in the laboratory
for determining the concentration of a substance
in a solution allowing a qualitative or quantitative
analysis of the sample.
Conventional spectrophotometer
• Upon passing of in mediums, light undergoes
a series of phenomena.
• Among these are featured reflection,
refraction, diffraction, absorption, diffusion,
polarization and other phenomena measured
by various instruments and devices.
The table below shows the wavelength ranges
used for carrying out spectrophotometry tests.
• The diagram in Figure 27 shows that the
incidental radiation [Io] can undergo a series of
transformations. It can be reflected [Ir],
transmitted [It], diffused [Id], absorbed and
directly emitted as fluorescence [If ]. The
phenomena on which spectrophotometry is
based are mainly absorption and transmission.
In order to understand how, it is necessary to
take Beer Lambert’s law into account.
Beer Lambert’s Law.
• it identifies the relationship between the concentration
of the sample and the intensity of light transmitted
through it.
• The transmittance [T] is the fraction of the incidental
light of determined wavelength passing through the
sample.
Where:
• It = intensity of the transmitted radiation
• Io = intensity of the incidental radiation
• The percentage of transmittance [%T] can be
expressed by the following equation:
• The concentration of light absorbing molecules in
a sample is proportional to the absorbance [A] of
that sample. It is expressed mathematically as:
• Where:
– A = Absorbance measured
– ε = Molecule absorbance coefficient [litres/moles/cm]
– l = Distance of the trajectory traversed (path length) by
the light in the sample
– c = Sample concentration [moles/litres]
• Absorbance [A] is related to transmittance [T]
through thefollowing equation:
Absorbance phenomenon
The graphs presented next demonstrate how absorbance
[A]and transmittance [T] vary as a function of the
concentration [C] according to Beer Lambert’s law.
Transmittance graph
Absorbance graph
• In conclusion it can be inferred that by increasing the concentration
of a substance, the transmittance is decreased and, upon increasing
the concentration of the substance, absorbance is increased.
• The linearity of Beer Lambert’s law is affected if the following
conditions occur.
– Displacement of the sample’s chemical balance as a function of the
concentration.
– Deviations in the absorbance coefficients, greater concentrations than
0.01 M due to electrostatic interaction between nearby molecules.
– Changes in the refraction index at high concentrations of the analyte.
– Diffusion of light due to particles in the sample.
– Fluorescence or phosphorescence of the sample.
– Non-monochromatic radiation.
SPECTROPHOTOMETER COMPONENTS
• The most important components of a spectrophotometer are the
following.
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The light source
The monochromator
The sample carrier
The detector system
The reading system
Light source
• Depending on the type of spectrophotometer, the light
source can be
– a tungsten lamp for visible light or
– a deuterium arc lamp for ultraviolet light.
– Some manufacturers have designed spectrophotometers
with long lasting xenon intermittent lamps emitting light in
the visible and ultraviolet ranges.
• The lamp(s) come factory-assembled on a base that
ensures a fixed position, to maintain optical
adjustment and focus when operating or when
replacing the bulb.
• The typical radiating energy emitted from a tungsten
lamp is between 2600 and 3000°K (Kelvin degrees).
Monochromator
• The monochomator is a set of elements used to disperse
white light into waves of different wavelengths, one of
which is used in the sample reading.
• In general, it has an entry crevice or groove which limits the
light radiation produced by the source and confines it to a
determined area;
• a set of mirrors for transmitting light through the optic
system; an element for separating the light radiation
wavelengths (which may be a prism or a diffraction (or
transmission) grating); and an exit opening for selecting the
wavelength required to illuminate the sample. Diffraction
gratings have the advantage of eliminating the non-linear
dispersion and being insensitive to changes in temperature.
Detector system
• The detection system can be designed with photocells,
phototubes, photodiodes or photomultipliers. This
depends on the ranges of wavelength, the sensitivity
and the required speed of response. The detection
system receives light from the sample and converts it
into an electrical signal proportional to the energy
received. This electrical signal can be processed and
amplifi ed to be interpreted by the reading system. A
summary of advantages and disadvantages of devices
normally used in detection systems is included in the
following table (see opposite).
Reading system
• The signal which leaves the detector goes through various
transformations. It is amplified and transformed until its
intensity becomes a proportional transmittance/absorbance
percentage. There are analogous reading systems (displaying
results on a reading scale) or digital ones (showing results on
a screen).
• Analogous indicators traditionally bear the name meters.
Their exactitude depends among other factors, on the length
and the number of divisions of the scale (the more divisions,
the more exact it is). Their main disadvantage is that they
can be incorrectly read, due to the operators’ fatigue or
errors identifying scales when there are several.
• Digital indicators usually show results on a screen as
illuminated alpha numerals. This makes reading errors less
likely.
Advantages and disadvantages of
common detection devices
INSTALLATION REQUIREMENTS
• For the correct functioning of a
spectrophotometer, the following is required:
– An electric supply source that complies with the
norms and standards used in the country. In
American countries, voltages of 110 V and
frequencies of 60 Hz are generally used. Other
parts of the World require 220-230V/50-60 Hz.
– A clean, dust free, environment.
– A stable work table away from equipment that
generate vibrations (centrifuges, agitators).
SPECTROPHOTOMETER
MAINTENANCE
• Routine maintenance required vary in complexity, ranging from
careful cleaning of components to specialized procedures carried
out by a trained specialized technician or engineer with the
technical information for different manufacturers’ models and
designs.
• Following manufacturer’s instructions and careful use will
guarantee a prolonged operational life. In recent models,
manufacturers have incorporated automatic routines of calibration
and verification.
• In this document general maintenance recommendations
applicable to a wide range of spectrophotometers are presented. It
is emphasized that specialized routines can only be performed
according to the specific manufacturer’s recommendations for each
particular model. General routine maintenance for a
spectrophotometer in good condition and the frequency of
estimated checks are as follows:
Inspection of the instrument’s
surroundings
• Frequency: Annually
– The area in which the spectrophotometer is
installed must be inspected visually and tested
electrically in order to guarantee the safety of the
operator.
– The inspection covers the electrical installation
and the installation area (physical infrastructure
related to the spectrophotometer).
Electrical installation
• It must be verified and tested for ensuring the
following:
– There is an electrical outlet or receptacle with a ground
pole.
– The receptacle is in good condition and is no further than
1.5 m from the spectrophotometer.
– The voltage is of an appropriate level and must not vary by
more than 5% of the voltage specified on the equipment’s
plate.
– The receptacle’s polarity is correct.
• These tests must be done by an electrical technician or
an engineer and results must be recorded to allow
follow-up over time.
Installation area
1. Check that there is free space around the spectrophotometer for
two purposes.
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First, for the connecting cables to pass without hindrances and for
other components or support equipment (e.g. the voltage
stabilizer).
Second, to allow adequate ventilation of the equipment when it is in
operation.
2. Test the integrity of the counter, its state and cleanliness.
3. Verify that there is no equipment installed that can transmit
vibrations in proximity. (E.g. centrifuges).
4. Verify that it is not affected by excessively humid conditions, dust
or high temperatures.
5. Avoid installing the equipment where it receives direct solar
radiation.
6. Do not install the equipment where there are magnetic fields or
intense electromagnetic radiation.
7. Ensure installation area is free from the influence of gases and
corrosive substances.
The most important aspects are:
1. Check that the work table supporting the spectrophotometer is in good condition.
2. Test the spectrophotometer. Verify that buttons or control switches and mechanical
closures are mounted firmly and that their identification labels are clear.
3. Ensure that accessories are clean, not showing cracks and that their functional state is
optimal.
4. Confirm that mechanical adjustment parts (nuts, screws, bolts, etc.) are adjusted and are
in good condition.
5. Check that electrical connectors do not have cracks or ruptures, that they are joined
correctly to the line.
6. Verify that cables are not showing signs of splicing, that they are not frayed and that they
do not have worn-out insulation.
7. Check that cables securing devices and terminals are free of dust, filth or corrosion.
These same cables must not be worn out or show signs of deterioration.
8. Check that the grounding system (internal and external) is standardized, of approved
type, functional and correctly installed.
9. Ensure that circuit switches or interrupters, the fuse box and indicators are free from
dust, filth and corrosion.
10. Check the external electrical components for signs of overheating.
General maintenance
Cleaning of spills
• In case of a leak in the sample holder or carrier, the
spill must be cleaned according to the following
procedure:
1. Turn off the spectrophotometer and disconnect the
cable from the electrical feed.
2. Use a syringe for cleaning the sample holder. Absorb
as much liquid that can possibly be extracted.
3. Dry the sample holder with a medicinal cotton bud.
4. Use lens paper or a clean piece of soft textured cloth
for cleaning the window of the photocell.
5. Clean the exterior of the instrument with a piece of
cloth moistened with distilled water.
Cleaning of quartz cuvettes
• To maintain quartz cuvettes in good condition:
– Wash the cuvettes using a diluted alkaline solution
such as NaOH 0.1 M and a diluted acid such as
HCl, 0.1 M.
– Rinse cuvettes several times with distilled water.
– Conduct rigorous and careful cleaning procedures
on cuvettes if samples used can deposit films.
– Some manufacturers recommend using special
detergents for cleaning cuvettes.
Battery changes
Of low battery indication appears on the instrument’s
screen.
• Turn off the spectrophotometer.
• Disconnect the electrical feed cable.
• Open the battery compartment and remove the worn out
batteries.
• Clean the electrical contact points.
• Install new batteries with the same specifications as the
originals.
• Close the compartment.
• Reconnect the equipment.
• Adjust the date and time information.
Change of bulb/lamp
• Common lamp change steps are as follows:
– Verify that the bulb is not functioning In modern equipment, a sign will
appear on the screen or an error code. In old equipment, the light will
simply no longer work.
– Turn off the spectrophotometer.
– Disconnect the feed cable.
– Undo the screws securing the top of the lamp’s compartment.
– Undo the screws keeping the lamp’s mechanism fixed.
– Undo the screws fastening the electrical connection cable to the lamp
– Install a new lamp with the same characteristics as the original. Use
gloves to avoid getting fingerprints on the surface of the lamp.
– Reconnect the electrical feed cables to the lamp.
– Reinstall the screws keeping the lamp in place.
– Replace the screws securing the lamp’s compartment’s cover.
– Reconnect the spectrophotometer.
– Turn the equipment ON and carry out the equipment’s recalibration
procedure stipulated by the manufacturer.
Preventive Maintenance
– Clean the spectrophotometer externally using a piece of fine cloth dampened with
distilled water.
– Inspect and clean the electrical feed cable.
– Verify that the lamp is clean and in good state.
– Check the protection fuse.
– Put the instrument in the operational configuration.
– Activate the “on” switch and allow it to warm up for five (5) minutes. Verify that:
• The lights or pilot indicators work.
• The reading indicators stay on zero (0).
• The light source works.
– Carry out an escaping current test in the “on” and “off ” position.
– Calibrate the front panel of the spectrophotometer according to the manufacturer’s
instructions.
– Measure the equipment’s sensitivity.
– Conduct a test according to Beer’s law.
– Return the spectrophotometer to the initial configuration .
GOOD PRACTICES WHEN USING THE
SPECTROPHOTOMETER
1. Calibrate the spectrophotometer every time a set of
samples is to be analyzed.
2. Keep the cover of the sample holder and
compartment closed during the measurement.
3. Avoid reusing disposable cuvettes.
4. Only use quartz cuvettes for carrying out analysis
under 310 nm.
5. Avoid the use of plastic cuvettes if using organic
solvents.
6. Use high quality boron silicate glassware for preparing
standards.
7. Avoid the use of sodium glass (sodium oxide)
7. Carefully clean the glass cuvettes after use.
8. Use high quality reagents. The diluents used
(water or solvents) must be free of impurities.
9. Verify that samples or standards did not degas
inside the cuvettes.
10. Take into account that not all substances
comply with Beer’s law. Carry out linearity
tests on the range of concentrations to be
used. It is recommended to prepare a group of
known high standard solutions and verify the
results.